Analysis of Escherichia coli taxonomy has expanded into a species-complex with the identification of divergent cryptic clades. A key question is the evolutionary trajectory of these clades and their relationship to isolates of clinical or veterinary importance. Since they have some environmental association, we screened a collection of E. coli isolated from a long-term spring barley field trial for their presence. While most isolates clustered into the enteric-clade, four of them clustered into Clade-V, and one in Clade-IV. The Clade -V isolates shared >96% intraclade average nucleotide sequence identity but <91% with other clades. Although pan-genomics analysis confirmed their taxonomy as Clade -V (E. marmotae), retrospective phylogroup PCR did not discriminate them correctly. Differences in metabolic and adherence gene alleles occurred in the Clade -V isolates compared to E. coli sensu scricto. They also encoded the bacteriophage phage-associated cyto-lethal distending toxin (CDT) and antimicrobial resistance (AMR) genes, including an ESBL, blaOXA-453. Thus, the isolate collection encompassed a genetic diversity, and included cryptic clade isolates that encode potential virulence factors. The analysis has determined the phylogenetic relationship of cryptic clade isolates with E. coli sensu scricto and indicates a potential for horizontal transfer of virulence factors.

Abstract Pectobacterium carotovorum has an incomplete Entner–Doudoroff (ED) pathway, including enzyme 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase (Eda) but lacking phosphogluconate dehydratase (Edd), while P. atrosepticum (Pba) has a complete pathway. To understand the role of the ED pathway in Pectobacterium infection, mutants of these two key enzymes, Δeda and Δedd, were constructed in Pba SCRI1039. Δeda exhibited significant decreased virulence on potato tubers and colonization in planta and was greatly attenuated in pectinase activity and the ability to use pectin breakdown products, including polygalacturonic acid (PGA) and galacturonic acid. These reduced phenotypes were restored following complementation with an external vector expressing eda. Quantitative reverse transcription PCR analysis revealed that expression of the pectinase genes pelA, pelC, pehN, pelW, and pmeB in Δeda cultured in pyruvate, with or without PGA, was significantly reduced compared to the wild type, while genes for virulence regulators (kdgR, hexR, hexA, and rsmA) remained unchanged. However, Δedd showed similar phenotypes to the wild type. To our knowledge, this is the first demonstration that disruption of eda has a feedback effect on inhibiting pectin degradation and that Eda is involved in building the arsenal of pectinases needed during infection by Pectobacterium.

As there is no agreed national list of species of socio-economic and/or cultural value for Scotland, we developed a set of criteria for selecting species. These include: • Species prioritised for conservation value • Species identified as being culturally important • Species providing important ecosystem services • Game species • Species collected for food or medicine Using these criteria, we selected 26 species for initial assessment. For each species, we produced a Genetic Scorecard, outlining: • Relevant genetic conservation issues for the species in question • The importance of its genetic diversity on an international scale • An evaluation of the genetic risks facing in situ populations • A statement of confidence in the assessment • The degree to which representation in ex situ collections mitigate against genetic diversity loss. • An overall ‘traffic light’ score of genetic risks and whether current conservation actions are effective Using 2010 as a baseline reference point, the approach assesses contemporary genetic issues, and likely future issues during a 25-year window from the point of assessment. For quantification of levels of risk, we adopted the following framework: • Negligible: No obviously detectable genetic problems occurring or expected over the next 25 years. • Moderate: Moderate genetic problems occurring or expected over the next 25 years; e.g.: - Moderate loss of populations that are likely to contain unique diversity (e.g., resulting in losses of up to 25% of important genetic types / distinct populations); - Clearly observable fitness problems in up to 25% of populations due to low genetic variation and subsequent inbreeding depression; - Marked and clearly observable loss of genetic integrity by hybridisation at up to 25% of populations; - Severe restrictions on regeneration/recruitment/reproduction in many or most populations of long-lived species limiting evolutionary change in the immediate future. • Serious: Serious genetic problems occurring or expected over the next 25 years; e.g.: - Severe loss of populations that are likely to contain unique diversity (e.g., resulting in losses of > 25% of important genetic types / distinct populations); - Loss of any highly divergent endemic lineages that are globally unique; - Strong, clearly observable fitness problems in >25% of populations due to low genetic variation and subsequent inbreeding depression; - Marked and clearly observable loss of genetic integrity by hybridisation at >25% of populations. The assessment is based on expert opinion, using direct genetic data, where available, combined with information on species biology, abundance and distribution. Where no direct genetic data are available, the genetic risk assessment is based on species biology, abundance and distribution.

Whole cell MALDI is regularly used for the identification of bacteria to species level in clinical Microbiology laboratories. However, there remains a need to rapidly characterize and differentiate isolates below the species level to support outbreak management. We describe the implementation of a modified preparative approach for MALDI-MS combined with a custom analytical computational pipeline as a rapid procedure for subtyping Shigatoxigenic E. coli (STEC) and accurately identifying strain-specifying biomarkers. The technique was able to differentiate E. coli O157:H7 from other STEC. Within O157 serotype O157:H7 isolates were readily distinguishable from Sorbitol Fermenting O157 isolates. Overall, nine homogeneous groups of isolates were distinguished, each exhibiting distinct profiles of defining mass spectra features. This offers a robust analytical tool useable in reference/diagnostic public health scenarios.

ISOPENTENYLTRANSFERASE (IPT) genes play important roles in the initial steps of cytokinin synthesis, exist in plant and pathogenic bacteria, and form a multigene family in plants. Protein domain searches revealed that bacteria and plant IPT proteins were to assigned to different protein domains families in the Pfam database, namely Pfam IPT (IPTPfam) and Pfam IPPT (IPPTPfam) families, both are closely related in the P-loop NTPase clan. To understand the origin and evolution of the genes, a species matrix was assembled across the tree of life and intensively in plant lineages. The IPTPfam domain was only found in few bacteria lineages, whereas IPPTPfam is common except in Archaea and Mycoplasma bacteria. The bacterial IPPTPfam domain miaA genes were shown as ancestral of eukaryotic IPPTPfam domain genes. Plant IPTs diversified into class I, class II tRNA-IPTs, and Adenosine-phosphate IPTs; the class I tRNA-IPTs appeared to represent direct successors of miaA genes were found in all plant genomes, whereas class II tRNA-IPTs originated from eukaryotic genes, and were found in prasinophyte algae and in euphyllophytes. Adenosinephosphate IPTs were only found in angiosperms. Gene duplications resulted in gene redundancies with ubiquitous expression or diversification in expression. In conclusion, it is shown that IPT genes have a complex history prior to the protein family split, and might have experienced losses or HGTs, and gene duplications that are to be likely correlated with the rise in morphological complexity involved in fine tuning cytokinin production.

The enzyme ISOPENTENYLTRANSFERASE (IPT) is responsible for the rate limiting step of cytokinin biosynthesis, an important plant hormone with key roles in meristem maintenance and organ development. In this study, we isolated IPT genes from the acaulescent Streptocarpus rexii, a plant that shows an unorthodox development starting with post-germination anisocotyly, in which cytokinins play an integral role. Three adenosine phosphate-IPTs and two tRNA-IPTs were isolated from S. rexii. Their expression levels and patterns in different tissues were compared by means of realtime-PCR and mRNA in-situ hybridization. We found that each SrIPT had a distinctive expression pattern. Interestingly, in vegetative tissues as well as in meristems only the adenosine phosphate-IPT SrIPT5 and the tRNA-IPT SrIPT9 were found. In addition, they were differentially affected by external hormone application, suggesting their different regulation and expression during meristem formation and maintenance and lamina growth. Our results indicate that SrIPTs are involved in shaping the architecture of S. rexii, working differentially and redundantly, and show that differentially expressed IPT genes regulate plant form.

aSummary: This R package helps to implement a robust approach to deal with mass spectrometry (MS) data. It is aimed at alleviating reproducibility issues and pernicious effects of deviating signals on both data pre-processing and downstream data analysis. Based on robust statistical methods, it facilitates the identification and filtering of low-quality mass spectra and atypical peak profiles as well as monitoring and data handling through pre-processing, which extends existing computational tools for high-throughput data. Availability and implementation: MALDIrppa is implemented as a package for the R environment for data analysis and it is freely available to download from the CRAN repository at https://CRAN.R-project.org/package=MALDIrppa. Contact: javier.palarea@bioss.ac.uk.

There is a significant knowledge gap with regard to non-filarial nematodes and their relationships, if any, with intracellular bacteria, with only sporadic reports in the literature. An intracellular bacteria Xiphinematobacter, belonging to subdivision 2 of the Verrucomicrobia, was previously reported in the ovaries of three species of the non-filarial Xiphinema americanum-group of nematodes. We explored the diversity of Xiphinematobacter in 22 populations of X. americanum sourced from six continents and conservatively have identified nine phylotypes, six of which have not previously been reported. A geographic basis to the phylotypes was noted with phylotypes A and B only found in Europe, whereas phylotypes F, G, H and I were mainly found in North America. Phylotypes C, D and E showed greater geographical variation. Sequences of Xiphinematobacter from this study help to inform the taxonomy of Verrucomicrobia such that the status and composition of Verrucomicrobia subdivision 2 potentially requires reflection.

Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-β-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-β-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.

An important component of barley cell walls, particularly in the endosperm, is (1,3;1,4)-β- glucan, a polymer that has proven health benefits in humans and that influences processability in the brewing industry. Genes of the cellulose synthase-like (Csl) F gene family have been shown to be involved in (1,3;1,4)-β-glucan synthesis but many aspects of the biosynthesis are still unclear. Examination of the sequence assembly of the barley genome has revealed the presence of an additional three HvCslF genes (HvCslF11, HvCslF12 and HvCslF13) which may be involved in (1,3;1,4)-β-glucan synthesis. Transcripts of HvCslF11 and HvCslF12 mRNA were found in roots and young leaves, respectively. Transient expression of these genes in Nicotiana benthamiana resulted in phenotypic changes in the infiltrated leaves, although no authentic (1,3;1,4)-β-glucan was detected. Comparisons of the CslF gene families in cereals revealed evidence of intergenic recombination, gene duplications and translocation events. This significant divergence within the gene family might be related to multiple functions of (1,3;1,4)-β-glucans in the Poaceae. Emerging genomic and global expression data for barley and other cereals is a powerful resource for characterising the evolution and dynamics of complete gene families. In the case of the CslF gene family, the results will contribute to a more thorough understanding of carbohydrate metabolism in grass cell walls.

In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species.

Background Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D). Methodology/Principal Findings Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae. Conclusions/Significance This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.

Background The potato genome sequence derived from the Solanum tuberosum Group Phureja clone DM1-3 516 R44 provides unparalleled insight into the genome composition and organisation of this important crop. A key class of genes that comprises the vast majority of plant resistance ( R ) genes contains a nucleotide-binding and leucine-rich repeat domain, and is collectively known as NB-LRRs. Results As part of an effort to accelerate the process of functional R gene isolation, we performed an amino acid motif based search of the annotated potato genome and identified 438 NB-LRR type genes among the ~39,000 potato gene models. Of the predicted genes, 77 contain an N-terminal toll/interleukin 1 receptor (TIR)-like domain, and 107 of the remaining 361 non-TIR genes contain an N-terminal coiled-coil (CC) domain. Physical map positions were established for 370 predicted NB-LRR genes across all 12 potato chromosomes. The majority of NB-LRRs are physically organised within 63 identified clusters, of which 50 are homogeneous in that they contain NB-LRRs derived from a recent common ancestor. Conclusions By establishing the phylogenetic and positional relationship of potato NB-LRRs, our analysis offers significant insight into the evolution of potato R genes. Furthermore, the data provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from Solanum species.

Summary: Data visualization can play a key role in comparative genomics, for example, underpinning the investigation of conserved synteny patterns. Strudel is a desktop application that allows users to easily compare both genetic and physical maps interactively and efficiently. It can handle large datasets from several genomes simultaneously, and allows all-by-all comparisons between these. Availability and implementation: Installers for Strudel are available for Windows, Linux, Solaris and Mac OS X at http://bioinf.scri.ac.uk/strudel/. Contact: strudel@scri.ac.uk; micha.bayer@scri.ac.uk

This study used PCR-RFLP to investigate the genetic variability of pmp-encoding genes from fifty-two Chlamydophila abortus (C. abortus) strains originating from abortion cases from various geographical regions and host species. Six primer pairs were used to PCR-amplify DNA fragments encoding eighteen pmps. PCR products were digested using four restriction endonucleases and Bayesian methodologies were used to compare RFLP profiles and assign strains to a RFLP genotype. Strains could be assigned to 2 genotypes in the region encoding pmp18D, 3 genotypes in the regions encoding pmp1A-pmp2B, pmp3E-pmp6H and pmp11G-pmp15G, 4 genotypes in the region encoding pmp7G-pmp10G and 5 genotypes in the region encoding pmp16G-pmp17G. In all regions, the majority of strains (88.4-96.1%) had the same genotype as the reference strain S26/3. No correlation could be made between genotype, host species or geographical origin except for the two variant Greek strains, LLG and POS, which formed a discrete genotype in all pmp-encoding regions except pmp18D. Relative rates of evolution calculated for each pmp-encoding gene locus suggest that differing selective pressures and functional constraints may exist on C. abortus polymorphic membrane proteins. These findings suggest that although intraspecies heterogeneity of pmp-encoding genes in C. abortus is low, the sequence heterogeneity should be an important consideration when using pmps as the basis for novel diagnostics or vaccine development. (C) 2011 Elsevier B.V. All rights reserved.

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the induction of protective immunity.

To the Editor: Despite the worldwide economic impact of, and welfare issues associated with, infectious bovine abortifacients, as well as potential zoonotic threats to human health, accurate diagnosis of the causes of abortion is uncommon (1). This poor diagnosis could be explained in part by the lack of identification of infectious abortifacient agents. Although Chlamydophila abortus is a known etiologic agent of ruminant abortion, several novel species of Chlamydia-like organisms have recently emerged as putative ruminant abortifacients. Waddlia chondrophila was isolated from the brain and nervous tissue of an aborted bovine fetus in Germany (2), and Parachlamydia acanthamoebae and other unidentified Chlamydia-like species were identified in 18.3% of bovine placenta samples in Switzerland (3,4). Given the paucity of information about the causes of infectious bovine abortion and the high prevalence of Chlamydia-like organisms in the samples from Switzerland, we attempted to determine whether such organisms can be detected in bovine fetal tissues in the United Kingdom. Pooled tissue samples comprising brain, heart, and/or placenta (depending on availability) were obtained from bovine fetuses submitted for diagnosis to the Scottish Agricultural College Disease Surveillance Centre, Dumfries, Scotland, UK, during 2008. Tissue pools were homogenized by using a Precellys bead mill homogenizer (Bertin Technologies, Ann Arbor, MI, USA), and DNA was extracted by using the Wizard Genomic DNA Purification Kit (Promega, Southampton, UK) according to manufacturer’s instructions. A pan-Chlamydiales PCR for the 16S rDNA sequence was performed by using forward primer 16S FOR2 (5′-CGT GGA TGA GGC ATG CAA GTC GA-3′) and reverse primer 16S REV2 (5′-CAA TCT CTC AAT CCG CCT AGA CGT CTT AG-3′) to generate amplicons of ≈260 bp (5). Negative-control reactions contained DNA-free water instead of extracted DNA. PCR products were purified (QIAquick PCR Purification Kit; QIAGEN, Crawley, UK) before direct sequencing by using the PCR primers and dideoxy chain termination/cycle sequencing on an ABI 3730XL DNA sequencer (MWG Operon, Ebersberg, Germany). After the initial PCR, 22 (26.5%) of the 83 fetal samples tested were Chlamydiales positive. Serologic, bacteriologic, and histopathologic examination of fetal tissues identified no other infectious abortifacient agents in the Chlamydiales-positive samples. Sequence information was successfully obtained for 15 of these 22 samples with forward and reverse primers; sequences ranged from 140 bp to 194 bp (European Molecular Biology Laboratory/GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"GQ919016-GQ919030","start_term":"GQ919016","end_term":"GQ919030","start_term_id":"884330660","end_term_id":"884330820"}}GQ919016-GQ919030). These 15 short sequences were carefully aligned to a representative set of 22 similar Chlamydiales 16S rDNA sequences, identified by a BLAST (www.ncbi.nlm.nih.gov/BLAST) similarity search of the European Molecular Biology Laboratory/GenBank database, plus alignment of an outgroup of 7 non-Chlamydiales sequences. A Bayesian phylogenetic tree (Markov Chain Monte Carlo settings: 2 runs of 625,000 generations; burn-in of 125,000 generations; trees sampled every 100 generations) was then estimated with a general time reversible + Γ nucleotide substitution model by using the MrBayes program (6) launched from the TOPALi v2 package (7). Despite the short sequence length of the 15 samples, the tree was well resolved with the Chlamydiales sequences and formed 3 clusters (Chlamydiaceae, Rhabdochlamydiaceae/Simkaniaceae, and Parachlamydiaceae/Waddliaceae/Criblamydiaceae) (Figure). Two of these sequence clusters represented 10 and 5 of the samples, whereas no samples were represented in the cluster containing the Chlamydiaceae, which includes C. abortus. Most (10/15) sequences were found in the cluster containing the Parachlamydiaceae. This finding agrees with those of the aborted bovine placenta studies in Switzerland (3,4) and provides further evidence that Parachlamydia-like species may play a substantial role in bovine abortion in mainland Europe and the United Kingdom. Four of the remaining 5 samples clustered with members of the family Rhabdochlamydiaceae; the fifth sequence (CLBUK3), although present in the same Rhabdochlamydiaceae/Simkaniaceae cluster, appeared to be more distinct from other family members. Figure Bayesian phylogenetic tree demonstrating the relationship of 15 isolated organisms from the older chlamydiales samples to known chlamydial species. Cand., Candidatus; R., Rhabdochlamydia; P., Protochlamydia. The identification of these organisms in such a large percentage of the bovine fetal tissue samples tested may indicate a role for these organisms in undiagnosed bovine abortions in the United Kingdom and Europe and may be a zoonotic source of infection for humans. Indeed, considerable evidence supports a role for Parachlamydia spp. in human pneumonia, whereas Rhabdochlamydia spp. is a suspected cause (8). In addition, evidence suggests that P. acanthamoebae crosses the human placenta to the unborn fetus (9). Also, the presence of both parachlamydial and rhabdochlamydial DNA in the lung secretions of hospitalized premature human neonates recently correlated with increased medical interventions and increased duration of hospital stay (10). We demonstrate the presence of Parachlamydiaceae and Rhabdochlamydiaceae species in bovine abortions in the United Kingdom. Given the zoonotic potential and the economic and welfare impacts of bovine abortion on the agricultural sector, further studies are required to understand the incidence and pathogenic roles of these organisms in both humans and animals. These studies should include broader molecular epidemiologic studies, as well as detailed histologic/immunohistochemical investigations and organism recovery through culture of infected placental and fetal tissues.

Summary: TOPALi v2 simplifies and automates the use of several methods for the evolutionary analysis of multiple sequence alignments. Jobs are submitted from a Java graphical user interface as TOPALi web services to either run remotely on high-performance computing clusters or locally (with multiple cores supported). Methods available include model selection and phylogenetic tree estimation using the Bayesian inference and maximum likelihood (ML) approaches, in addition to recombination detection methods. The optimal substitution model can be selected for protein or nucleic acid (standard, or protein-coding using a codon position model) data using accurate statistical criteria derived from ML co-estimation of the tree and the substitution model. Phylogenetic software available includes PhyML, RAxML and MrBayes. Availability: Freely downloadable from http://www.topali.org for Windows, Mac OS X, Linux and Solaris. Contact: iain. milne@scri. ac. uk